| Budget Amount *help |
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Outline of Final Research Achievements |
This cap-dependent endonuclease activity termed as cap snatching may provide a unique target for novel anti-influenza viral agents. For the screening of candidate inhibitors for cap-snatching activity, it is essential to establish a method to efficiently produce Cap1-RNA substrate and a convenient assay system for cap-snatching activity. A short 3’-biotinylated RNA oligonucleotide was prepared by an in vitro transcription system utilizing T7 RNA polymerase. Purified 3’-biotinylated RNA oligonucleotide by C18 cartridge column was subjected to a sequential capping reaction with recombinant enzymes of vaccinia virus D1R containing with RNA 5’-triphosphatase and mRNA guanylyltransferase, yeast
ABD1 as (guanine-N7)-methyltransferase, and VP39 as (nucleoside-2'-O-)-methyltransferase. Cap-snatching activity could be analyzed a pull-down assay based on the streptavidin-biotin interaction, yielding results consistent with those obtained by polyacrylamide gel electrophoresis.
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